SINEs are the most abundant of the mammalian mobile elements. In humans, Alu elements make up about 11% of the mass of the genome. We estimate that there is about 1 new Alu insertion in every 200 humans born. These insertions cause about 0.1% of all human germ-line disease and could possibly contribute to somatic cell damage involved in cancer progression or aging. A secondary consequence of Alu insertions is the creation of homologous sequences interspersed throughout the genome that can contribute to unequal homologous recombination. These recombination events contribute to another 0.3% of human germ line disease, and are almost certainly a major factor in LOH involved in cancer progression. SiNEs are considered non-autonomous mobile elements in that they have no coding capacity and therefore must parasitize the retrotransposition apparatus from other elements or cellular functions. There is growing evidence that SINEs may be largely, or wholly, dependent on the retrotransposition enzymes supplied by mammalian Li elements. The SINE elements are able to make transcripts via transcription by RNA polymerase III. This proposal focuses on several cis and trans components of the SINE retroposition process and ultimately designs an assay for detection of Alu retroposition in culture. The trans elements considered are the proteins that bind with SiNEs to form ribonucleoprotein particles (RNPs). Although we have no data to suggest whether the specific influences will be direct, indirect, positive or negative, these RNPs almost certainly influence the retrotransposition process, as well as any other impacts SINEs may have on their host organism. We have demonstrated polyA-binding protein (PABP) as a component of SINE complexes. Thus, we are beginning to be able to predict how the RNP may be able to interact with other cellular components and how that might influence SINEs in the cell. We still need to identify the other protein components of the SINE RNP to provide a better picture of SINE RNA behavior. We have been able to recreate a complex with the same mobility in a native mobility shift assay that allows us to carry out a rapid and effective purification of the binding proteins. We will use this assay to isolate and characterize the SINE RNA binding proteins. We will also utilize ehimaeric and mutant RNA-expressing constructs to determine the domains of the RNA associated with different protein bindings, and characterize the expression patterns and nature of each of the RNP proteins. The importance of poly A tails as a primary cis component is highlighted by an evolutionary analysis that suggests that the active source Alu elements have exceptionally long A tails encoded in the genome. We will further define this analysis to determine whether it is a useful tool for identifying source elements, as well as very recently inserted Alus. All of this information will be combined with an anchored allele-specific PCR assay we have developed for detecting new Alu inserts, or an alternative in situ hybridization strategy, to create a tissue culture system for Alu retroposition. This system will allow a detailed analysis of the factors with influence the SINE retroposition process.